Effects Of Intermittent High Glucose On Proliferation,the Expression Of Apoptosis-associated Proteins In Culturerat Vascular Smooth Muscle Cells

Objective: Vascular complications are the leading cause of death in diabetic patients, atherosclerosis is accelerated by the coexistence of diabetes mellitus. Poor control of diabetes is associated with the development of micro- and macroangiopathy, hyperglycemia in diabetic patients is believed to play a major role in the development of these vascular complications. Increased proliferation of vascular smooth muscle cells (VSMCs) is a key feature in the atherosclerotic lesion. It is well established that cell growth is a fundamental feature of intimal hyperplasia, and it is widely accepted that perturbations in the regulation of apoptosis are equally important. Several findings support the concept that hyperglycemia accelerates the development of atherosclerosis. High concentration of glucose enhances growth rate in cultured VSMCs. Treatment with a high glucose not only significantly attenuate apoptosis in response to serum withdrawal in cultured rat VSMCs, but also markedly increase the expression of Bcl-2. Another report showed that there was a significant decrease in the DNA fragmentation ratio of human coronary artery smooth muscle cells cultured at high glucose concentration. Excessive accumulation of VSMCs in atherosclerosis suggests reduced apoptosis and excessive cell proliferation in the lesions. The pathological effects exist in two ways: chronic intermittent hyperglycemia and chronic persistent hyperglycemia. Chronic intermittent hyperglycemia means astatus of chronic intermittent or paroxysmal high level of blood glucose,which is also named as drift of glucose. Recent studies have found that the former one has the riority to accelerate the happening and development of macrovascular complications in patients with DM,which has captured the attention of experts in the field of the prevention and treatment of macroangiopathy. In vitro studies found that,hyperglycemia can induce apoptosis of vascular endothelial cell,however intermittent hyperglycemia can lead to higher apoptosis ratio of the cultivated human vascular endothelial cell. Therefore,intermittent hyperglycemia is more dangerous than relative stable hyperglycemia. Fluctuating hyperglycemia may accelerate the advancement of atherosclerosis.The current study aimed at the impact of intermittent high concentration of glucose and constant high concentration of glucose on proliferation and apoptosis of rat vascular smooth muscle cell line A7r5 and the effects on expression of apoptosis-associated protein Bcl-2 and Bax,and the impossible mechanisms were discussed.Methods: Vascular smooth muscle cell lines A7r5 was purchased from CCTCC(China Center for Type Culture Collection). Cultures were maintained at 37℃in which the A7r5 cells were thawed as soon as possible. Then they were inoculated with DMEM culture media at 37℃in air containing 5% CO2. Cells were inoculated in 50 ml culture bottles and on 96 proe culture boards for 48 hours.The above cells were inculated in another media with 0% FBS(fetal bovine serum) for 24 hours to make the cells in phase G0/Gl and then divided into three groups: (1) Continuous normal glucose medium (5.5 mmol/L). (2) Continuous high glucose (25 mmol/L). (3) High glucose (25 mmo/L) 3 h normal glucose (5.5 mmo/L) 2 h and media were alternated three times a day,then mantained with high glucose (25 mmol/L) at night. After 24 hours. Its survival rate was detected by MTT assay and cell cycle distribution was analyzed by flow cytometry. The levels of Bcl-2 and Bax protein expressions were measured by western blot.Results:1 MTT assays were used to characterize the viability of VSMCs . Intermittent high glucose and constant high glucose conditions have higher cell survival rates than that in the normal concentraion condition(0.205±0.017, 0.186±0.011 vs 0.172±0.009 respectively P<0.001)The proliferating viability of VSMCs induced by intermittent high glucose was remarkably increased when compared with the high glucose group (P<0.01).2 To detect the cell cycle progression of VSMCs, flow cytometric analysis was performed. The percentage of cells in S phase in high concertration glucose group,intermittent high concentration glucose group and normale on certration glucose group is 37.57±1.54 %, 41.92±1.76 %, 34.00±1.45 % respectively. Compared with normal glucose, intermittent high glucose significantly increased the percentage of cells in the S phase. (P<0.01). Also, the percentage of cells in the S phase in intermittent high glucose group was much higher than that in constant high glucose group (P<0.05). Also, the percentage of cells in the S phase in intermittent high concentration glucose group was much higher than that in constant high glucose group . Although it seemed like that constant high glucose increased the percentage of cells in the G0/G1 phase compared with normal glucose, the changes were not significant (P>0.05).3 Western blot. The Bcl-2 protein expression and Bcl-2/Bax ratio are higher in the group of constant high glucose than that in the group of normal glucose(P <0.01).Compared with the intermittent high concentration glucose group, Bcl-2 protein expression in constant high glucose group is lower(P <0.01).Conclusion:1 Compared with the normal glucose,constant high glucose and intermittent high glucose have stonger stimula-tory effects on proliferation of VSMCs,and the effect of intermittent high glucose on proliferation is more than constant high glucose.2 After 24 h exposure,compared with constant high glucose, intermittent high glucose decreased the percentage of cells in the G0/G1 phase, increased the S phase. Intermittent high glucose induced the proliferation of VSMCs which may due to the promotion of the cell cycle.3 Compared with the constant high glucose,intermittent high glucose can increase the expression of apoptosis-associated proteinBcl-2 and Bcl-2/Bax ratio . 4 For the first time we proved that compared with constant high glucose, intermittent high glucose has more stimulatory effects on the proliferation in VSMCs, inhibit apoptosis through the regulation of apoptosis-ralated proteins, therefore leading to the acceleration of onset and development of atherosclerosis in diabetes.